Development of a preventive vaccine for filovirus infection in primates

ABSTRACT

The present invention relates generally to viral vaccines and, more specifically, to filovirus vaccines and methods of eliciting an immune response against a filovirus or disease caused by infection with filovirus.

This application is a division of U.S. patent application Ser. No.10/491,121, filed Aug. 23, 2004, which is a 371 national phase ofPCT/US02/30251, filed Sep. 24, 2002, which claims priority to U.S.Provisional Patent Application No. 60/326,476, filed Oct. 1, 2001, thecontents of both are incorporated herein in the entirety for allpurposes.

FIELD OF THE INVENTION

The present invention relates generally to viral vaccines and, moreparticularly, to filovirus vaccines and methods of eliciting an immuneresponse against a filovirus or a disease caused by infection withfilovirus.

BACKGROUND OF THE INVENTION

The Ebola viruses, and the genetically-related Marburg virus, arefiloviruses associated with outbreaks of highly lethal hemorrhagic feverin humans and primates in North America, Europe, and Africa (Peters, C.J. et al. in: Fields Virology, eds. Fields, B. N. et al. 1161-1176,Philadelphia, Lippincott-Raven, 1996; Peters, C. J. et al. 1994 SeminVirol 5:147-154). Ebola viruses are negative-stranded RNA virusescomprised of four subtypes, including those described in the Zaire,Sudan, Reston, and Ivory Coast episodes (Sanchez, A. et al. 1996 PNASUSA 93:3602-3607). Although several subtypes have been defined, thegenetic organization of these viruses is similar, each containing sevenlinearly arrayed genes. Among the viral proteins, the envelopeglycoprotein exists in two alternative forms, a 50-70 kilodalton (kDa)secreted protein of unknown function encoded by the viral genome and a130 kDa transmembrane glycoprotein generated by RNA editing thatmediates viral entry (Peters, C. J. et al. in: Fields Virology, eds.Fields, B. N. et al. 1161-1176, Philadelphia, Lippincott-Raven, 1996;Sanchez, A. et al. 1996 PNAS USA 93:3602-3607). Other structural geneproducts include the nucleoprotein (NP), matrix proteins VP24 and VP40,presumed nonstructural proteins VP30 and VP35, and the viral polymerase(reviewed in Peters, C. J. et al. in: Fields Virology, eds. Fields, B.N. et al. 1161-1176, Philadelphia, Lippincott-Raven, 1996). Althoughspontaneous variation of its RNA sequence does occur in nature, thereappears to be less nucleotide polymorphism within Ebola subtypes thanamong other RNA viruses (Sanchez, A. et al. 1996 PNAS USA 93:3602-3607),suggesting that immunization may be useful in protecting against thisdisease. Previous attempts to elicit protective immune responses againstEbola virus using traditional active and passive immunization approacheshave, however, not succeeded in primates (Peters, C. J. et al. in:Fields Virology, eds. Fields, B. N. et al. 1161-1176,

Philadelphia, Lippincott-Raven, 1996; Clegg, J. C. S. et al. 1997 NewGeneration Vaccines, eds.: Levine, M. M. et al. 749-765, New York, N.Y.Marcel Dekker, Inc.; Jahrling, P. B. et al. 1996 Arch Virol Suppl11:135-140). It would thus be desirable to provide a vaccine to elicitan immune response against a filovirus or disease caused by infectionwith filovirus. It would further be desirable to provide methods ofmaking and using said vaccine.

SUMMARY OF THE INVENTION

Outbreaks of hemorrhagic fever caused by the Ebola virus are associatedwith high mortality rates that are a distinguishing feature of thishuman pathogen. The highest lethality is associated with the Zairesubtype, one of four strains identified to date (Feldmann, H. et al.1994 Virology 199:469-473; Sanchez, A. et al. 1996 PNAS USA93:3602-3607). Its rapid progression allows little opportunity todevelop natural immunity, and there is currently no effective anti-viraltherapy. Therefore, vaccination offers a promising intervention toprevent infection and limit spread. Here we describe a highly effectivevaccine strategy for Ebola virus infection in primates. A combination ofDNA immunization and boosting with adenoviral vectors that encode viralproteins generated cellular and humoral immunity in cynomolgus macaques.Challenge with a lethal dose of the highly pathogenic, wild-type, 1976Mayinga strain of Ebola Zaire virus resulted in uniform infection incontrols, who progressed to a moribund state and death in less than oneweek. In contrast, all vaccinated animals were asymptomatic for morethan six months, with no detectable virus after the initial challenge.These findings demonstrate that it is possible to develop a preventivevaccine against Ebola virus infection in primates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows VRC6000 (pVR1012-GP(Z)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 2 shows VRC6001 (pVR1012x/s-GP(Z)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 3 shows VRC6002 (pVR1012-GP(Z) delta MUC) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 4 shows VRC6003 (pVR1012-GP(Z) delta MUC delta FUR) construct map(see, Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses inTable 2).

FIG. 5 shows VRC6004 (pVR1012-GP(Z) delta GP2) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 6 shows VRC6005 (pVR1012-GP(Z) delta GP2 delta C-term A) constructmap (see, Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses inTable 2).

FIG. 7 shows VRC6006 (pVR1012-GP(Z) delta GP2 delta C-term B) constructmap (see, Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses inTable 2).

FIG. 8 shows VRC6007 (pVR1012-GP(Z) delta GP2 delta FUS) construct map(see, Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses inTable 2).

FIG. 9 shows VRC6008 (pVR1012-GP(Z) delta TM) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 10 shows VRC 6052 (pVR1012-GP(Z) delta SGP) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 11 shows VRC 6101 (pVR1012x/s Ebola GP(R) (dTM)) construct map(see, Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses inTable 2).

FIG. 12 shows VRC 6110 (pAdApt Ebola GP(R) (dTM)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 13 shows VRC6200 (pVR1012-GP(S)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 14 shows VRC 6201 (pVR1012x/s Ebola GP(S)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 15 shows VRC6202 (pVR1012-GP(S) delta TM) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 16 shows VRC6300 (pVR1012-GP(IC)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 17 shows VRC6301 (pVR1012x/s-GP(IC)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 18 shows VRC6302 (pVR1012-GP(IC) delta TM) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 19 shows VRC6303 (pVR1012x/s Ebola GP (IC) (dTM)) construct map(see, Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses inTable 2).

FIG. 20 shows VRC6310 (pAdApt Ebola GP (IC) (dTM)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 21 shows VRC6351 (pVR1012x/s-SGP(IC)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 22 shows VRC6400 (pVR1012-NP) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 23 shows VRC6401 (pVR1012x/s-NP) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 24 shows VRC6500 (pVR1012-VP35) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 25 shows VRC6600 (pAD/CMV-GP(dTM)(Z-CITE-S) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 26 shows VRC6601 (pAdApt Ebola GP(S)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 27 shows VRC6602 (pAdApt Ebola GP(S)(dTM)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 28 shows VRC6603 (pAdApt Ebola GP(Z)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 29 shows VRC6604 (pAdApt Ebola GP(Z)(dTM)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 30 shows VRC6701 (pVR1012-Marburg) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 31 shows VRC6702 (pVR1012x/s Marburg GP (dTM)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 32 shows VRC6710 (pAdApt Marburg GP (dTM)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 33 shows VRC6800 (pVR1012x/s Lassa GP) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 34 shows VRC6801 (pVR1012x/s Lassa GP (dTM) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 35 shows VRC6810 (pAdApt Lassa GP) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 36 shows VRC6811 (pAdApt Lassa GP (dTM)) construct map (see,Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses in Table 2).

FIG. 37 shows CMV/R Ebola GP (Z) deltaTM/h (codon optimized) constructmap (see, Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses inTable 2).

FIG. 38 shows pVR1012 Ebola GP (Z, P87666) delta TM/h (codon optimized)construct map (see, Ebola/Marburg/Lassa Plasmids, and RecombinantAdenoviruses in Table 2).

FIG. 39 shows CMV/R Ebola GP (S/Gulu) delta TM/h (codon optimized)construct map (see, Ebola/Marburg/Lassa Plasmids, and RecombinantAdenoviruses in Table 2).

FIG. 40 shows CMV/R Ebola GP (S,Q66798) delta TM/h (codon optimized)construct map (see, Ebola/Marburg/Lassa Plasmids, and RecombinantAdenoviruses in Table 2).

FIG. 41 shows VRC6802, pVR1012x/s Lassa delta TM/h (codon optimized)construct map (see, Ebola/Marburg/Lassa Plasmids, and RecombinantAdenoviruses in Table 2).

FIG. 42 shows VRC6703, pVR1012x/s Marburg delta TM/h (codon optimized)construct map (see, Ebola/Marburg/Lassa Plasmids, and RecombinantAdenoviruses in Table 2).

FIG. 43 shows CMV/R Ebola NP construct map (see, Ebola/Marburg/LassaPlasmids, and Recombinant Adenoviruses in Table 2).

FIG. 44 is a diagrammatic representation of secreted glycoprotein (SGP)and glycoprotein (GP) molecules of Ebola virus (Zaire species isolatedin 1976) showing important structural features. The white N-terminalregions of SGP and GP correspond to identical (shared) sequences, whilethe black C termini identify sequences unique to GP or SGP molecules.The common signalase cleavage sites for both SGP and GP and the furincleavage site for GP0 (uncleaved form of GP) (↓) were determined byN-terminal sequencing. Also shown are cysteine residues (S), predictedN-linked glycosylation sites (Y-shaped projections), a predicted fusionpeptide, a heptad repeat sequence, and a transmembrane anchor sequence.In Ebola viruses, the positions of these structures are conserved andtheir sequences are very similar or, in the case of N-linkedglycosylation sites, are at least concentrated in the central region ofGP. Signalase cleavage site is SEQ ID NO: 48, Furin cleavage site is SEQID NO: 49, and Fusion peptide is SEQ ID NO: 50.

FIG. 45 is a diagrammatic representation of the structural GP. Shown isthe predicted orientation of the GP1-GP2 heterodimer linked byundetermined disulfide bonding (indicated by the question mark).Intramolecular disulfide bonds that are shown come from priorpredictions based on similarities to retrovirus glycoprotein structures.See FIG. 44 for other features of the amino acid sequence.

FIG. 46 shows induction of the cytopathic effects by Ebola virusglycoproteins and mapping of the molecular determinants ofcytopathicity.

FIG. 47 shows Ebola-specific antibody responses generated by differentDNA/adenovirus prime-boost combinations. Data are the means of thereciprocal endpoint dilution for each group of mice and error barsrepresent the standard deviation.

FIG. 48 (A-D) shows DNA-Adenovirus immunization of cynomolgus macaques.A) Immunization schedule for DNA and/or adenovirus injections, andchallenge with the wild-type Mayinga strain of the Zaire subtype ofEbola virus. B) Elisa titers of Ebola-specific antibodies in serum.Serum was collected at week 12 (open bar) and 2 days before theimmunization at week 24 (closed bar). C) Lymphoproliferative responsesto Ebola-secreted glycoprotein (SGP) following immunization. Barsrepresent the average fold-proliferation of all four blood samples foreach subject. The standard deviation is not shown because the baselinelevel of induction varied between experiments. However, PBMC from all 8animals were assayed within the same experiment for each time point, andthe averages displayed in the figure are representative of the resultsobtained for any single time point. D) Lymphoproliferative responses toEbola SGP in bulk PBMC following depletion of lymphocyte subsets. PBMCfrom week 24 were treated with Dynal magnetic beads coated with theindicated antibody to deplete CD4⁺ or CD8⁺ cell subsets. Cells remainingafter depletion were normalized for input cell number and stimulated asdescribed in the Example. Results are shown for two control (Subjects 2and 3) and two vaccinated (Subjects 6 and 7) monkeys.

FIG. 49 (A-C) shows protection of cynomolgus macaques against lethalchallenge with Ebola virus after DNA-adenovirus immunization. A, B)Hepatic enzyme levels in monkeys after challenge with Ebola virus. Liverenzymes [alanine aminotransferase (ALT) and aspartate aminotransferase(AST)] levels in the non-human primate sera were measured by standardrecommended procedures using General chemistry 12 reagent disk for thePiccolo™ Analyzer (Abaxis, Inc., Sunnyvale, Calif.). Results are shownfor four immunized (closed symbols) and four control (open symbols)monkeys. C) Plasma viraemia in monkeys following infection with Ebolavirus. Crosses represent time of death in control animals [days 5(subject 1) and 6 (subjects 2 and 4)]. One control animal, subject 3,was euthanized on day 7 when it was moribund. One vaccinated animal thatwas resistant to infection, subject 5, was euthanized on day 10 forhistological examination of tissues. By day 17, none of the animals haddetectable viraemia, and they remained aviraemic for the duration of theobservation period (6 months). Data are the reciprocal endpoint dilutionof serum for each monkey. Results are shown for four immunized (closedsymbols) and four control (open symbols) monkeys.

FIG. 50 (A-B) shows enhanced expression of modified CMV expressionvector, CMV/R.

FIG. 51 shows enhanced immunogenicity of modified CMV expression vector,CMV/R, in mice.

TABLE 1 Ebola/Marburg/Lassa GenBank Accession Numbers. Gene GenBankAccession number Ebola Zaire GP U23187, P87666 Ebola Zaire NP J04337Ebola Sudan GP U28134, Q66798 Ebola Sudan NP AF173836 Ebola Ivory CoastGP U28006 Ebola Ivory Coast NP JO4336 Ebola Reston GP U23152 EbolaReston NP Marburg GP Z12132 Marburg NP X68495 Lassa GP AF181853 Lassa NPAF246121

TABLE 2 Ebola/Marburg/Lassa Plasmids, and Recombinant Adenoviruses SEQConstruct Construct Name/Description Construct Map Name ID NO FigureVRC6000 VRC6000 (pVR1012-GP(Z)) pVR1012-GP(Z) 1 1 VRC6001 VRC6001(pVR1012x/s-GP(Z)) pVR1012x/s Ebola GP(Z) 2 2 VRC6002 VRC6002(pVR1012-GP(Z) delta MUC) pVR1012-GP(Z) delta MUC 3 3 VRC6003 VRC6003(pVR1012-GP(Z) delta MUC delta FUR) pVR1012-GP(Z) delta MUC delta FUR 44 VRC6004 VRC6004 (pVR1012-GP(Z) delta GP2) pVR1012-GP(Z) delta GP2 5 5VRC6005 VRC6005 (pVR1012-GP(Z) delta GP2 delta C-term A) pVR1012-GP(Z)delta GP2 delta C-term A 6 6 VRC6006 VRC6006 (pVR1012-GP(Z) delta GP2delta C-term B) pVR1012-GP(Z) delta GP2 delta C-term B 7 7 VRC6007VRC6007 (pVR1012-GP(Z) delta GP2 delta FUS) pVR1012-GP(Z) delta GP2delta FUS 8 8 VRC6008 VRC6008 (pVR1012-GP(Z) delta TM) pVR1012-GP(Z)delta TM 9 9 VRC6052 VRC 6052 (pVR1012-GP(Z) delta SGP) pVR1012-GP(Z)delta SGP 10 10 VRC6101 VRC 6101 (pVR1012x/s Ebola GP(R) (dTM))pVR1012x/s Ebola GP(R)(dTM) 11 11 VRC6110 VRC 6110 (pAdApt Ebola GP(R)(dTM)) pAdApt Ebola GP(R) (dTM) 12 12 VRC6200 VRC6200 (pVR1012-GP(S))pVR1012-GP(S) 13 13 VRC6201 VRC 6201 (pVR1012x/s Ebola GP(S)) pVR1012x/sEbola GP(S) 14 14 VRC6202 VRC6202 (pVR1012-GP(S) delta TM) pVR1012-GP(S)delta TM 15 15 VRC6300 VRC6300 (pVR1012-GP(IC)) pVR1012-GP(IC) 16 16VRC6301 VRC6301 (pVR1012x/s-GP(IC)) pVR1012x/s Ebola GP(IC) 17 17VRC6302 VRC6302 (pVR1012-GP(IC) delta TM) pVR1012-GP(IC) delta TM 18 18VRC6303 VRC 6303 (pVR1012x/s Ebola GP (IC) (dTM)) pVR1012x/s EbolaGP(IC)(dTM) 19 19 VRC6310 VRC 6310 (pAdApt Ebola GP (IC) (dTM)) pAdAptEbola GP(IC)(dTM) 20 20 VRC6351 VRC6351 (pVR1012x/s-sGP(IC))pVR1012x/s-sGP(IC) 21 21 VRC6400 VRC6400 (pVR1012-NP) pVR1012-NP 22 22VRC6401 VRC6401 (pVR1012x/s-NP) pVR1012x/s Ebola-NP 23 23 VRC6500VRC6500 (pVR1012-VP35) pVR1012-VP35 24 24 VRC6600 VRC6600(pAD/CMV-GP(dTM)(Z-CITE-S) pAD/CMV-GP(dTM)(Z-CITE-S) 25 25 VRC6601VRC6601 (pAdApt Ebola GP(S)) pAdApt Ebola GP(S) 26 26 VRC6602 VRC 6602(pAdApt Ebola GP(S)(dTM)) pAdApt Ebola GP(S)(dTM) 27 27 VRC6603 VRC6603(pAdApt Ebola GP(Z)) pAdApt Ebola GP(Z) 28 28 VRC6604 VRC 6604 (pAdAptEbola GP(Z)(dTM)) pAdApt Ebola GP(Z)(dTM) 29 29 VRC6701 VRC6701(pVR1012-Marburg) pVR1012 Marburg 30 30 VRC6702 VRC 6702 (pVR1012x/sMarburg GP (dTM)) pVR1012x/s Marburg GP(dTM) 31 31 VRC6710 VRC 6710(pAdApt Marburg GP (dTM)) pAdApt Marburg GP (dTM) 32 32 VRC6800 VRC6800(pVR1012x/s Lassa GP) pVR1012x/s Lassa GP 33 33 VRC6801 VRC6801(pVR1012x/s Lassa GP (dTM) pVR1012x/s Lassa GP (dTM) 34 34 VRC6810VRC6810 (pAdApt Lassa GP) pAdApt Lassa GP 35 35 VRC6811 VRC6811 (pAdAptLassa GP (dTM)) pAdApt Lassa GP (dTM) 36 36 CMV/R Ebola GP (Z) deltaTM/h(codon optimized) CMV/R Ebola GP(Z) delta TM/h 37 37 pVR1012 EbolaGP(Z,P87666)delta TM/h (codon optimized) pVR1012x/s Ebola GP(Z) delta TM/h(P87666) 38 38 CMV/R Ebola GP (S/Gulu) delta TM/h (codon optimized)CMV/R-GP(S/G)(deltaTM)/h 39 39 CMV/R Ebola GP (S, Q66798) delta TM/h(codon optimized) CMV/R-GP(S, Q66798)(dTM)/h 40 40 VRC6802 VRC6802,pVR1012x/s Lassa delta TM/h (codon optimized) pVR1012x/s Lassa (codonoptimized) 41 41 VRC6703 VRC6703, pVR1012x/sMarburgdeltaTM/h (codonoptimized) PVR1012x/s Marburg (codon optimized) 42 42 CMV/R Ebola NPCMV/R Ebola NP 43 43

DETAILED DESCRIPTION OF THE INVENTION

Filovirus vaccines are provided comprising a nucleic acid moleculeencoding a filoviral structural protein operatively-linked to a controlsequence in a pharmaceutically acceptable excipient. In one embodiment,the nucleic acid molecule encodes the transmembrane form of the viralglycoprotein (GP). In another embodiment, the nucleic acid moleculeencodes the secreted form of the viral glycoprotein (SGP). In yetanother embodiment, the nucleic acid molecule encodes the viralnucleoprotein (NP).

The present invention further includes vaccines comprising nucleic acidmolecules encoding filoviral structural proteins other than GP, SGP, andNP, e.g., other structural gene products which elicit an immune responseagainst a filovirus or disease caused by infection with filovirus. Thenucleic acid molecules of the vaccines of the present invention encodestructural gene products of any Ebola viral strain including the Zaire,Sudan, Ivory Coast and Reston strains. Nucleic acid molecules encodingstructural gene products of the genetically-related Marburg virusstrains may also be employed. Moreover, the nucleic acid molecules ofthe present invention may be modified, e.g., the nucleic acid moleculesset forth herein may be mutated, as long as the modified expressedprotein elicits an immune response against a pathogen or disease. Forexample, the nucleic acid molecule may be mutated so that the expressedprotein is less toxic to cells. The present invention also includesvaccines comprising a combination of nucleic acid molecules. Forexample, and without limitation, nucleic acid molecules encoding GP, SGPand NP of the Zaire, Sudan and Ivory Coast Ebola strains may be combinedin any combination, in one vaccine composition.

The present invention also provides methods for immunizing a subjectagainst disease caused by infection with filovirus comprisingadministering to the subject an immunoeffective amount of a filovirusvaccine. Methods of making and using filovirus vaccines are alsoprovided by the present invention including the preparation ofpharmaceutical compositions.

Biochemical Analysis of Secreted and Virion Glycoproteins of EbolaVirus.

Ebola (EBO) viruses are members of the Filoviridae and cause a severe,often fatal form of hemorrhagic fever disease in human and/or non-humanprimates. The glycoprotein (GP) gene of filoviruses is the fourth gene(of seven) from the 3′ end of the negative-strand RNA genome. All EBOviruses characterized thus far have the same unconventional type of GPgene organization that results in the expression of a secreted,nonstructural glycoprotein (SGP) in preference to the structural GP. TheSGP is encoded in a single frame (0 frame), while the GP is encoded intwo frames (0 and −1 frames). Expression of the GP occurs when the twoframes are connected through a transcriptional editing event thatresults in the insertion of a single extra adenosine (added to a run ofseven adenosines).

Referring to FIG. 44, for Zaire species of EBO virus, the N-terminal 295residues (including signal sequence) of the SGP (364 total residues) andGP (676 total residues) are identical, but the length and composition oftheir C-terminal sequences are unique. The GP, a type 1 transmembraneprotein, is found on the surface of the infectious virion and functionsin attachment structure in the binding and entry of the virus intosusceptible cells. Comparisons of GP predicted amino acid sequences forall species of EBO virus show a general conservation in the N-terminaland C-terminal regions (each approximately one-third of the totalsequence) and are separated by a highly variable middle section. Thisprotein is highly glycosylated, containing large amounts of N- andO-linked glycans, and for Marburg (MBG) virus (another type offilovirus) has been shown to form trimers. Just N terminal to thetransmembrane anchor sequence of the GP (residues 650 to 672) is a motif(residues 585 to 609) that is highly conserved in filoviruses. Thissequence also has a high degree of homology with a motif in theglycoproteins of oncogenic retroviruses that has been shown to beimmunosuppressive in vitro. Partially overlapping this motif is a heptadrepeat sequence (53 residues; positions 541 to 593) that is thought tofunction in the formation of intermolecular coiled coils in the assemblyof trimers, similar to structures predicted for the surfaceglycoproteins of other viruses. Immediately N terminal to this sequenceis a predicted fusion peptide followed closely by a putative multibasiccleavage site for a subtilisin/kexin-like convertase, furin. Cleavage byfurin has been indirectly demonstrated by use of specific inhibitors andis predicted to occur at the last arginine in the sequence RRTRR↓(position 501 from the beginning of the open reading frame [ORF]).Although the role of the SGP is less defined, recent studies have shownthat SGP can bind to neutrophils, while GP binds to endothelial cells.The different binding patterns of SGP and GP suggest that despite havingidentical N-terminal amino acid sequences (˜280 residues), theseglycoproteins are structurally very distinct from one another.

Referring to FIG. 45, the glycoproteins expressed by a Zaire species ofEbola virus were analyzed for cleavage, oligomerization, and otherstructural properties to better define their functions. The 50- to70-kDa secreted and 150-kDa virion/structural glycoproteins (SGP and GP,respectively), which share the 295 N-terminal residues, are cleaved nearthe N terminus by signalase. A second cleavage event, occurring in GP ata multibasic site (RRTRR↓) (SEQ ID NO: 51) that is likely mediated byfurin, results in two glycoproteins (GP1 and GP2) linked by disulfidebonding. This furin cleavage site is present in the same position in theGPs of all Ebola viruses (R[R/K]X[R/K]R↓), and one is predicted forMarburg viruses (R[R/K]KR↓), although in a different location. Based onthe results of cross-linking studies, investigators were able todetermine that Ebola virion peplomers are composed of trimers of GP1-GP2heterodimers and that aspects of their structure are similar to those ofretroviruses (including lentiviruses like HIV-1 and HIV-2),paramyxoviruses, and influenza viruses. Investigators also determinedthat SGP is secreted from infected cells almost exclusively in the formof a homodimer that is joined by disulfide bonding.

Referring to FIG. 46, investigators defined the main viral determinantof Ebola virus pathogenicity; synthesis of the virion glycoprotein (GP)of Ebola virus Zaire induced cytotoxic effects in human endothelialcells in vitro and in vivo. This effect mapped to aserine-threonine-rich, mucin-like domain of this type I transmembraneglycoprotein, one of seven gene products of the virus. Gene transfer ofGP into explanted human or porcine blood vessels caused massiveendothelial cell loss within 48 hours that led to a substantial increasein vascular permeability. Deletion of the mucin-like region of GPabolished these effects without affecting protein expression orfunction. GP derived from the Reston strain of virus, which causesdisease in non-human primates but not in man, did not disrupt thevasculature of human blood vessels. In contrast, the Zaire GP inducedendothelial cell disruption and cytotoxicity in both non-human primateand human blood vessels, and the mucin domain was required for thiseffect. These findings indicate that GP, through its mucin domain, isthe viral determinant of Ebola pathogenicity and likely contributes tohemorrhage during infection.

Nucleic Acid Molecules

As indicated herein, nucleic acid molecules of the present invention maybe in the form of RNA or in the form of DNA obtained by cloning orproduced synthetically. The DNA may be double-stranded orsingle-stranded. Single-stranded DNA or RNA may be the coding strand,also known as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

Nucleic acid molecules of the present invention include DNA moleculescomprising an open reading frame (ORF) encoding a wild-type filovirusstructural gene product; and DNA molecules which comprise a sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode an ORF of a wild-typefilovirus structural gene product. Of course, the genetic code is wellknown in the art.

The present invention is further directed to fragments of the nucleicacid molecules described herein. By a fragment of a nucleic acidmolecule having the nucleotide sequence of an ORF encoding a wild-typefilovirus structural gene product is intended fragments at least about15 nt., and more preferably at least about 20 nt., still more preferablyat least about 30 nt., and even more preferably, at least about 40 nt.in length. Of course, larger fragments 50, 100, 150, 200, 250, 300, 350,400, 450, or 500 nt. in length are also intended according to thepresent invention as are fragments corresponding to most, if not all, ofthe nucleotide sequence of the ORF encoding a wild-type filovirusstructural gene product. By a fragment at least 20 nt. in length, forexample, is intended fragments which include 20 or more contiguous basesfrom the nucleotide sequence of the ORF of a wild-type filovirusstructural gene product.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of thefilovirus structural protein. In particular, such nucleic acid fragmentsof the present invention include nucleic acid molecules encodingepitope-bearing domains of a filovirus structural protein, where thedomain is the GP/SGP identity domain, the mucin-like domain, the furincleavage site, the fusion peptide domain, the heptad repeat domain, thetransmembrane anchor domain, and the intracellular domain, and anycombination thereof, for example, a filovirus glycoprotein having atruncation at the carboxy terminus to delete the transmembrane anchorand intracellular domain, a filovirus glycoprotein having a truncationat the carboxy terminus to delete the heptad repeat domain andtransmembrane anchor and intracellular domain, a filovirus glycoproteinhaving a truncation at the carboxy terminus to delete the fusion peptidedomain, heptad repeat domain, and transmembrane anchor and intracellulardomain, a filovirus glycoprotein having a truncation at the carboxyterminus to delete the furin cleavage site, fusion peptide domain,heptad repeat domain, and transmembrane anchor and intracellular domain,a filovirus glycoprotein having a truncation at the carboxy terminus todelete the mucin-like domain, furin cleavage site, fusion peptidedomain, heptad repeat domain, and transmembrane anchor and intracellulardomain. Another example is a filovirus glycoprotein having an amino,internal, or carboxy deletion to delete the mucin-like domain, the furincleavage site, the fusion peptide domain, the heptad repeat domain, thetransmembrane anchor domain, or the intracellular domain.

In another aspect, the invention provides a nucleic acid moleculecomprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above. By “stringenthybridization conditions” is intended overnight incubation at 42° C. ina solution comprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt.), and more preferably at least about 20 nt.,still more preferably at least about 30 nt., and even more preferablyabout 30-70 nt. of the reference polynucleotide.

By a portion of a polynucleotide of “at least 20 nt. in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide. Of course, apolynucleotide which hybridizes only to a poly A sequence or acomplementary stretch of T (or U) residues, would not be included in apolynucleotide of the invention used to hybridize to a portion of anucleic acid of the invention, since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly A stretch orthe complement thereof (e.g., practically any double-stranded cDNAclone).

As indicated herein, nucleic acid molecules of the present inventionwhich encode a filovirus structural gene product may include, but arenot limited to those encoding the amino acid sequence of the full-lengthpolypeptide, by itself, the coding sequence for the full-lengthpolypeptide and additional sequences, such as those encoding a leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence,the coding sequence of the full-length polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, for example,ribosome binding and stability of mRNA; and additional coding sequencewhich codes for additional amino acids, such as those which provideadditional functionalities.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the filovirus structural gene product. Variants may occurnaturally, such as a natural allelic variant. By an “allelic variant” isintended one of several alternate forms of a gene occupying a givenlocus on a genome of an organism. (Genes II, Lewin, B., ed., John Wiley& Sons, 1985 New York). Non-naturally occurring variants may be producedusing art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions, which may involve one or more nucleotides. Thevariants may be altered in coding regions, non-coding regions, or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of thefilovirus structural gene product or portions thereof. Also especiallypreferred in this regard are conservative substitutions.

Further embodiments of the invention include nucleic acid moleculescomprising a polynucleotide having a nucleotide sequence at least 95%identical, and more preferably at least 96%, 97%, 98% or 99% identicalto a nucleotide sequence encoding a polypeptide having the amino acidsequence of a wild-type filovirus structural gene product or fragmentthereof or a nucleotide sequence complementary thereto.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a filovirusstructural gene product is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the Ebolavirus structural gene product. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to the reference nucleotidesequence can be determined conventionally using known computer programssuch as the Bestfit program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homologyalgorithm of Smith and Waterman, 1981 Advances in Applied Mathematics2:482-489, to find the best segment of homology between two sequences.When using Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence and that gapsin homology of up to 5% of the total number of nucleotides in thereference sequence are allowed.

The present application is directed to nucleic acid molecules at least95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences shownherein in the Sequence Listing which encode a polypeptide having Ebola,Marburg, or Lassa virus polypeptide activity. By “a polypeptide havingEbola, Marburg, or Lassa virus polypeptide activity” is intendedpolypeptides exhibiting Ebola, Marburg, or Lassa virus polypeptideactivity in a particular biological assay. For example, GP, SGP or NPprotein activity can be measured for changes in immunological characterby an appropriate immunological assay.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%, or99% identical to a nucleic acid sequence shown herein in the SequenceListing will encode a polypeptide “having Ebola, Marburg, or Lassa viruspolypeptide activity”. In fact, since degenerate variants of thesenucleotide sequences all encode the same polypeptide, this will be clearto the skilled artisan even without performing the above describedcomparison assay. It will be further recognized in the art that, forsuch nucleic acid molecules that are not degenerate variants, areasonable number will also encode a polypeptide having Ebola, Marburg,or Lassa virus polypeptide activity. This is because the skilled artisanis fully aware of amino acid substitutions that are either less likelyor not likely to significantly effect protein function (e.g., replacingone aliphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al. 1990 Science247:1306-1310, wherein the authors indicate that proteins aresurprisingly tolerant of amino acid substitutions.

Polypeptides and Fragments

The invention further provides a filovirus polypeptide having the aminoacid sequence encoded by an open reading frame (ORF) of a wild-typefilovirus structural gene, or a peptide or polypeptide comprising aportion thereof (e.g., SGP).

It will be recognized in the art that some amino acid sequences of thefilovirus polypeptides can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity.

Thus, the invention further includes variations of the filoviruspolypeptide which show substantial filovirus polypeptide activity orwhich include regions of filovirus protein such as the protein portionsdiscussed below. Such mutants include deletions, insertions, inversions,repeats, and type substitutions. As indicated, guidance concerning whichamino acid changes are likely to be phenotypically silent can be foundin Bowie, J. U. et al. 1990 Science 247:1306-1310.

Thus, the fragment, derivative or analog of the polypeptide of theinvention may be (i) one in which one or more of the amino acid residuesare substituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues include asubstituent group, or (iii) one in which additional amino acids arefused to the mature polypeptide, such as an IgG Fc fusion region peptideor leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table A).

TABLE A Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Ionizable: Acidic Aspartic Acid Glutamic AcidIonizable: Basic Arginine Histidine Lysine Nonionizable Polar AsparagineGlutamine Selenocystine Serine Threonine Nonpolar (Hydrophobic) AlanineGlycine Isoleucine Leucine Proline Valine Sulfur Containing CysteineMethionine

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions for any givenfilovirus polypeptide will not be more than 50, 40, 30, 20, 10, 5 or 3.

Amino acids in the filovirus polypeptides of the present invention thatare essential for function can be identified by methods known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham & Wells 1989 Science 244:1081-1085). The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as changes in immunological character.

The polypeptides of the present invention are conveniently provided inan isolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell or a native source. Forexample, a recombinantly produced version of the filovirus polypeptidecan be substantially purified by the one-step method described in Smithand Johnson 1988 Gene 67:31-40.

The polypeptides of the present invention include a polypeptidecomprising a polypeptide having the amino acid sequence of a wild-typefilovirus structural gene product or portion thereof or encoded by anucleic acid sequence shown herein in the Sequence Listing; as well aspolypeptides which are at least 95% identical, and more preferably atleast 96%, 97%, 98%, or 99% identical to those described above.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of an filoviruspolypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the filovirus polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least95%, 96%, 97%, 98%, or 99% identical to a reference amino acid sequencecan be determined conventionally using known computer programs such theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

In another aspect, the invention provides portions of the polypeptidesdescribed herein with at least 30 amino acids and more preferably atleast 50 amino acids. Preferred portions of the present inventioninclude polypeptides comprising an epitope-bearing portion of afilovirus structural protein. In particular, preferred portions of thepresent invention include polypeptides comprising an epitope-bearingdomain of a filovirus structural protein, where the domain is the GP/SGPidentity domain, the mucin-like domain, the furin cleavage site, thefusion peptide domain, the heptad repeat domain, the transmembraneanchor domain, and the intracellular domain, and any combinationthereof, for example, a filovirus glycoprotein having a truncation atthe carboxy terminus to delete the transmembrane anchor andintracellular domain, a filovirus glycoprotein having a truncation atthe carboxy terminus to delete the heptad repeat domain andtransmembrane anchor and intracellular domain, a filovirus glycoproteinhaving a truncation at the carboxy terminus to delete the fusion peptidedomain, heptad repeat domain, and transmembrane anchor and intracellulardomain, a filovirus glycoprotein having a truncation at the carboxyterminus to delete the furin cleavage site, fusion peptide domain,heptad repeat domain, and transmembrane anchor and intracellular domain,and a filovirus glycoprotein having a truncation at the carboxy terminusto delete the mucin-like domain, furin cleavage site, fusion peptidedomain, heptad repeat domain, and transmembrane anchor and intracellulardomain. Another example is a filovirus glycoprotein having an amino,internal, or carboxy deletion to delete the mucin-like domain, the furincleavage site, the fusion peptide domain, the heptad repeat domain, thetransmembrane anchor domain, or the intracellular domain.

The polypeptides of the invention may be produced by any conventionalmeans (Houghten, R. A. 1985 PNAS USA 82:5131-5135). The “SimultaneousMultiple Peptide Synthesis (SMPS)” process is described in U.S. Pat. No.4,631,211 to Houghten et al. (1986).

The present invention also relates to vectors which include the nucleicacid molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof filovirus polypeptides or fragments thereof by recombinanttechniques.

The present invention relates to “prime and boost” immunization regimesin which the immune response induced by administration of a primingcomposition is boosted by administration of a boosting composition. Thepresent invention is based on the inventors' experimental demonstrationthat effective boosting can be achieved using replication-defectiveadenovirus vectors, following priming with any of a variety of differenttypes of priming compositions. The present invention employsreplication-deficient adenovirus which, as the experiments describedbelow show, has been found to be an effective means for providing aboost to an immune response primed to antigen using any of a variety ofdifferent priming compositions.

Replication-deficient adenovirus derived from human serotype 5 has beendeveloped as a live viral vector by Graham and colleagues (Graham &Prevec 1995 Mol Biotechnol 3:207-20; Bett et al. 1994 PNAS USA91:8802-6). Adenoviruses are non-enveloped viruses containing a lineardouble-stranded DNA genome of around 3600 bp. Recombinant viruses can beconstructed by in vitro recombination between an adenovirus genomeplasmid and a shuttle vector containing the gene of interest togetherwith a strong eukaryotic promoter, in a permissive cell line whichallows viral replication. High viral titres can be obtained from thepermissive cell line, but the resulting viruses, although capable ofinfecting a wide range of cell types, do not replicate in any cellsother than the permissive line, and are therefore a safe antigendelivery system. Recombinant adenoviruses have been shown to elicitprotective immune responses against a number of antigens includingtick-borne encephalitis virus NS1 protein (Jacobs et al. 1992 J Virol66:2086-95) and measles virus nucleoprotein (Fooks et al. 1995 Virology210:456-65).

Remarkably, the experimental work described below demonstrates that useof embodiments of the present invention allows for recombinantreplication-defective adenovirus expressing an antigen to boost animmune response primed by a DNA vaccine. The replication-defectiveadenovirus was found to induce an immune response after intramuscularimmunization. In prime/boost vaccination regimes thereplication-defective adenovirus is also envisioned as being able toprime a response that can be boosted by a different recombinant virus orrecombinantly produced antigen.

Non-human primates immunized with plasmid DNA and boosted withreplication-defective adenovirus were protected against challenge. Bothrecombinant replication-deficient adenovirus and plasmid DNA arevaccines that are safe for use in humans. Advantageously, the inventorsfound that a vaccination regime used intramuscular immunization for bothprime and boost can be employed, constituting a general immunizationregime suitable for inducing an immune response, e.g., in humans.

The present invention in various aspects and embodiments employs areplication-deficient adenovirus vector encoding an antigen for boostingan immune response to the antigen primed by previous administration ofthe antigen or nucleic acid encoding the antigen.

A general aspect of the present invention provides for the use of areplication-deficient adenoviral vector for boosting an immune responseto an antigen.

One aspect of the present invention provides a method of boosting animmune response to an antigen in an individual, the method includingprovision in the individual of a replication-deficient adenoviral vectorincluding nucleic acid encoding the antigen operably linked toregulatory sequences for production of antigen in the individual byexpression from the nucleic acid, whereby an immune response to theantigen previously primed in the individual is boosted.

An immune response to an antigen may be primed by genetic immunization,by infection with an infectious agent, or by recombinantly producedantigen.

A further aspect of the invention provides a method of inducing animmune response to an antigen in an individual, the method comprisingadministering to the individual a priming composition comprising theantigen or nucleic acid encoding the antigen and then administering aboosting composition which comprises a replication-deficient adenoviralvector including nucleic acid encoding the antigen operably linked toregulatory sequences for production of antigen in the individual byexpression from the nucleic acid.

A further aspect provides for use of a replication-deficient adenoviralvector, as disclosed, in the manufacture of a medicament foradministration to a mammal to boost an immune response to an antigen.Such a medicament is generally for administration following prioradministration of a priming composition comprising the antigen.

The priming composition may comprise any viral vector, includingadenoviral, or other than adenoviral, such as a vaccinia virus vectorsuch as a replication-deficient strain such as modified virus Ankara(MVA) (Mayr et al. 1978 Zentralbl Bakteriol 167:375-90; Sutter and Moss1992 PNAS USA 89:10847-51; Sutter et al. 1994 Vaccine 12:1032-40) orNYVAC (Tartaglia et al. 1992 Virology 118:217-32), an avipox vector suchas fowlpox or canarypox, e.g., the strain known as ALVAC (Kanapox,Paoletti et al. 1994 Dev Biol Stand 1994 82:65-9), or a herpes virusvector.

The priming composition may comprise DNA encoding the antigen, such DNApreferably being in the form of a circular plasmid that is not capableof replicating in mammalian cells. Any selectable marker should not beresistant to an antibiotic used clinically, so for example Kanamycinresistance is preferred to Ampicillin resistance. Antigen expressionshould be driven by a promoter which is active in mammalian cells, forinstance the cytomegalovirus immediate early (CMV IE) promoter.

In particular embodiments of the various aspects of the presentinvention, administration of a priming composition is followed byboosting with first and second boosting compositions, the first andsecond boosting compositions being the same or different from oneanother, e.g., as exemplified below. Still further boosting compositionsmay be employed without departing from the present invention. In oneembodiment, a triple immunization regime employs DNA, then adenovirus(Ad) as a first boosting composition, and then MVA as a second boostingcomposition, optionally followed by a further (third) boostingcomposition or subsequent boosting administration of one or other orboth of the same or different vectors. Another option is DNA then MVAthen Ad, optionally followed by subsequent boosting administration ofone or other or both of the same or different vectors.

The antigen to be included in respective priming and boostingcompositions (however many boosting compositions are employed) need notbe identical, but should share epitopes. The antigen may correspond to acomplete antigen in a target pathogen or cell, or a fragment thereof.Peptide epitopes or artificial strings of epitopes may be employed, moreefficiently cutting out unnecessary protein sequence in the antigen andencoding sequence in the vector or vectors. One or more additionalepitopes may be included, for instance epitopes which are recognized byT helper cells, especially epitopes recognized in individuals ofdifferent HLA types.

Within the replication-deficient adenoviral vector, regulatory sequencesfor expression of the encoded antigen will include a promoter. By“promoter” is meant a sequence of nucleotides from which transportationmay be initiated of DNA operably linked downstream (i.e. in the 3′direction on the sense strand of double-stranded DNA). “Operably linked”means joined as part of the same nucleic acid molecule, suitablypositioned and oriented for transcription to be initiated from thepromoter. DNA operably linked to a promoter is “under transcriptionalinitiation regulation” of the promoter. Other regulatory sequencesincluding terminator fragments, polyadenylation sequences, enhancersequences, marker genes, internal ribosome entry site (IRES) and othersequences may be included as appropriate, in accordance with theknowledge and practice of the ordinary person skilled in the art: see,for example, Molecular Cloning: a Laboratory Manual, 2^(nd) edition,Sambrook et al. 1989 Cold Spring Harbor Laboratory Press. Many knowntechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, Ausubel et al. eds., John Wiley & Sons, 1994.

Suitable promoters for use in aspects and embodiments of the presentinvention include the cytomegalovirus immediate early (CMV IE) promoter,with or without intron A, and any other promoter that is active inmammalian cells.

Either or both of the priming and boosting compositions may include anadjuvant or cytokine, such as alpha-interferon, gamma-interferon,platelet-derived growth factor (PDGF), granulocyte macrophage-colonystimulating factor (GM-CSF) granulocyte-colony stimulating factor(gCSF), tumor necrosis factor (TNF), epidermal growth factor (EGF),IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12, or encoding nucleic acidtherefor.

Administration of the boosting composition is generally weeks or monthsafter administration of the priming composition, preferably about 2-3weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or28 weeks, or 32 weeks.

Preferably, administration of priming composition, boosting composition,or both priming and boosting compositions, is intramuscularimmunization.

Intramuscular administration of adenovirus vaccines or plasmid DNA maybe achieved by using a needle to inject a suspension of the virus orplasmid DNA. An alternative is the use of a needless injection device toadminister a virus or plasmid DNA suspension (using, e.g., Biojector™)or a freeze-dried powder containing the vaccine (e.g., in accordancewith techniques and products of Powderject), providing for manufacturingindividually prepared doses that do not need cold storage. This would bea great advantage for a vaccine that is needed in rural areas of Africa.

Adenovirus is a virus with an excellent safety record in humanimmunizations. The generation of recombinant viruses can be accomplishedsimply, and they can be manufactured reproducibly in large quantities.Intramuscular administration of recombinant replication-deficientadenovirus is therefore highly suitable for prophylactic or therapeuticvaccination of humans against diseases which can be controlled by animmune response.

The individual may have a disease or disorder such that delivery of theantigen and generation of an immune response to the antigen is ofbenefit or has a therapeutically beneficial effect.

Most likely, administration will have prophylactic aim to generate animmune response against a pathogen or disease before infection ordevelopment of symptoms.

Diseases and disorders that may be treated or prevented in accordancewith the present invention include those in which an immune response mayplay a protective or therapeutic role.

Components to be administered in accordance with the present inventionmay be formulated in pharmaceutical compositions. These compositions maycomprise a pharmaceutically acceptable excipient, carrier, buffer,stabilizer or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material may depend on the route of administration, e.g.,intravenous, cutaneous or subcutaneous, intramucosal (e.g., gut),intranasal, intramuscular, or intraperitoneal routes.

As noted, administration is preferably intradermal, subcutaneous orintramuscular.

Liquid pharmaceutical compositions generally include a liquid carriersuch as water, petroleum, animal or vegetable oils, mineral oil orsynthetic oil. Physiological saline solution, dextrose or othersaccharide solution or glycols such as ethylene glycol, propylene glycolor polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilizers, buffers,antioxidants and/or other additives may be included, as required.

A slow-release formulation may be employed.

Following production of replication-deficient adenoviral particles andoptional formulation of such particles into compositions, the particlesmay be administered to an individual, particularly human or otherprimate.

Administration may be to another mammal, e.g., rodent such as mouse, rator hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey,dog or cat.

Administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g., decisions ondosage etc., is within the responsibility of general practitioners andother medical doctors, or in a veterinary context a veterinarian, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 16^(th) edition, Osol, A. ed., 1980.

In one preferred regimen, DNA is administered (preferablyintramuscularly) at a dose of 10 micrograms to 50 milligrams/injection,followed by adenovirus (preferably intramuscularly) at a dose of5×10⁷-1×10¹² particles/injection.

The composition may, if desired, be presented in a kit, pack ordispenser, which may contain one or more unit dosage forms containingthe active ingredient. The kit, for example, may comprise metal orplastic foil, such as a blister pack. The kit, pack, or dispenser may beaccompanied by instructions for administration.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Delivery to a non-human mammal need not be for a therapeutic purpose,but may be for use in an experimental context, for instance ininvestigation of mechanisms of immune responses to an antigen ofinterest, e.g., protection against disease.

Further aspects and embodiments of the present invention will beapparent to those of ordinary skill in the art, in view of the abovedisclosure and following experimental exemplification, included by wayof illustration and not limitation, and with reference to the attachedfigures.

Development of a Preventive Vaccine for Ebola Virus Infection inPrimates

Genetic immunization has been shown to influence both humoral andcellular immune activation pathways and to protect against infection byhuman pathogens (Tang, D. C. et al. 1992 Nature 356:152-154; Ulmer, J.B. et al. 1993 Science 259:1745-1749; Wang, B. et al. 1993 PNAS USA90:4156-4160; Sedegah, M. et al. 1994 PNAS USA 91:9866-9870). Theeffectiveness of plasmid vaccines is thought to result from host cellprotein synthesis and endogenous presentation of the immunogen, andpossibly to immunostimulatory effects of plasmid DNA itself (Krieg, A.M. et al. 1995 Nature 374:546-549; Sato, Y. et al. 1996 Science273:352-354). DNA vaccines have been shown to elicit specific immuneresponses to Ebola virus antigens and to protect guinea pigs (Xu, L. etal. 1998 Nat Med 4:7-42) and mice (Vanderzanden, L. et al. 1998 Virology246:134-144) against challenge with Ebola virus adapted to producelethal infection in rodents (Connolly, B. M. et al. 1999 J Infect Dis179:S203-S217; Bray, M. et al. 1998 J Infect Dis 178:651-661). Althoughboth cell-mediated and humoral immune responses were elicited, antibodytiter correlated with the degree of protection in animals immunized withplasmids encoding proteins from the Zaire subtype of Ebola virus.

A broadly effective vaccine would need to provide immunity to themultiple Ebola subtypes isolated in human infections (Zaire, Sudan andIvory Coast), but a multivalent vaccine might dilute the specific immuneresponse demonstrated for the single subtype vaccine. To address thisconcern, we analyzed the efficacy of the original Ebola Zaire DNAvaccine in comparison to its use in combination with DNA from Ebolasubtypes Sudan and Ivory Coast. As in a previous study (Xu, L. et al.1998 Nat Med 4:7-42), immunization with a single plasmid encoding Zairesubtype virion glycoprotein, GP(Z), generated a substantialvirus-specific antibody response and conferred protective immunity inguinea pigs (Table I). Inclusion of a plasmid expressing Ebolanucleoprotein, NP, did not affect the antibody titer to Ebola GP(Z) ordiminish its protective efficacy. Further broadening of the vaccinecomponents to include NP and three subtypes of Ebola glycoprotein,Zaire, Ivory Coast and Sudan, GP(Z,IC,S)+NP, yielded a pre-challengeimmune response comparable to the single-plasmid vaccine. Moreover,complete protection from infection with Ebola Zaire was achieved inguinea pigs that received the multivalent vaccine (Table I, subjects13-16). Anamnestic antibody was not induced by the virus challenge,indicating that the vaccine itself provided an immune responsesufficient to efficiently clear the virus. These findings show thatmultivalent plasmid immunization did not substantially diminishglycoprotein (GP)-specific antibody production and its protectiveefficacy in a rodent model.

TABLE I Multivalent genetic immunization in guinea pigs ID ImmunizationELISA IgG Survival 1 Plasmid 0 No 2 Plasmid 0 No 3 Plasmid 0 No 4Plasmid 0 No 5 GP(Z) 6400 Yes 6 GP(Z) 6400 Yes 7 GP(Z) 6400 Yes 8 GP(Z)3200 Yes 9 GP(Z) + NP 6400 Yes 10 GP(Z) + NP 6400 Yes 11 GP(Z) + NP 6400Yes 12 GP(Z) + NP 6400 Yes 13 GP(Z, IC, S) + NP 6400 Yes 14 GP(Z, IC,S) + NP 1600 Yes 15 GP(Z, IC, S) + NP 6400 Yes 16 GP(Z, IC, S) + NP 6400Yes Table I. Comparison of multivalent vs. monovalent geneticimmunization in guinea pigs. Guinea pigs were immunized intramuscularlythree times at two-week intervals with 100 μg of DNA (Plasmid, 100 μgp1012; GP(Z), 100 μg pGP(Z); GP(Z) + NP, 75 μg pGP(Z) and 25 μg pNP;GP(Z, IC, S) + NP, 25 μg each of pGP(Z), pGP(IC), pGP(S) and pNP). Serumwas collected 6 weeks after the first injection and pre-challenge titersfor antibody to Ebola GP (ELISA IgG) were measured by ELISA (Ksiazek, T.G. et al. 1992 J Clin Microbiol 30: 947-950) and are displayed as thereciprocal endpoint dilution. Three months after the final immunizationthe animals were challenged as described (Xu, L. et al. 1998 Nat Med 4:37-42).

Because protection in the rodent model of Ebola virus infectioncorrelated with antibody titers, and efficient humoral responses mayinfluence clinical outcome in human disease (Baize, S. et al. 1999 NatMed 5:423-426; Maruyama, T. et al. 1999 J Virol 73:6024-6030), weconsidered it important to elicit a strong humoral response for vaccinestested in primates, although cell-mediated immunity is coordinatelyinduced and likely contributes to protection (Xu, L. et al. 1998 Nat Med4:37-42). Recently, regimens of DNA priming followed by administrationof viral vectors have demonstrated enhanced immune responses compared tovaccines using DNA alone (Sedegah, M. et al. 1998 PNAS USA 95:7648-7653;Hanke, T. et al. 1998 Vaccine 16:439-445; Robinson, H. L. et al. 1999Nat Med 5:526-534; Schneider, J. et al. 1998 Nat Med 4:397-402).Recombinant, replication-deficient adenoviruses can be grown to hightiter, infect antigen-presenting cells, and induce potent immuneresponses (Davis, A. R. et al. 1985 PNAS USA 82:7560-7564; Natuk, R. J.et al. 1992 PNAS USA 89:7777-7781; Xiang, Z. Q. et al. 1996 Virology219:220-227). Adenoviruses have shown a boosting effect in mice (Xiang,Z. Q. et al. 1999 J Immunol 162:6716-6723), but the combination of DNAand adenovirus has not been tested for efficacy in an infectiouschallenge model, and the success of this approach in primates is yetunknown. We therefore developed a recombinant adenoviral vector thatdirects high level GP expression ADV-GP(Z) and used this vector to testwhether a modified prime-boost strategy would augment the antibodyresponse to Ebola virus obtained with naked DNA alone. Mice wereinjected with DNA and adenovirus vectors either singly or incombinations, and cell-mediated and humoral immune responses wereassessed. A 10- to 100-fold increase in antibody titer was found in miceinjected with DNA followed by an adenovirus boost, compared to DNAimmunization alone (FIG. 47). An increase in cytotoxic T cell responseswas also observed with this combination. Immunization with ADV-GP(Z)alone yielded antibody titers that were not significantly different fromthose obtained with the DNA prime, adenovirus boost immunization. Thesedata suggest that immunogenicity of the Ebola GP DNA vaccine in mice isimproved by boosting with recombinant adenovirus and that this strategymight represent a useful approach to enhance immune responses innon-human primates.

Whereas the rodent model has been useful in the development of a vaccinestrategy, Ebola virus isolated directly from humans must first beadapted by multiple, sequential passage in rodents in order to produce alethal infection in mice or guinea pigs (Connolly, B. M. et al. 1999 JInfect Dis 179:S203-S217; Bray, M. et al. 1998 J Infect Dis178:651-661). Primate models of Ebola infection are thought to have astronger predictive value for human disease and immune protection. Wetherefore conducted studies in non-human primates using a bimodalDNA/ADV vaccine and the multiple plasmid strategy that correlated withprotection in guinea pigs. Cynomolgus macaques (Macaca fascicularis)received 3 injections of naked DNA vectors at 4-week intervals (FIG.48A) and, after several months of rest which has been shown to boostimmune responses (Letvin, N. L. et al. 1997 PNAS USA 94:9378-9383), wereboosted with recombinant adenovirus expressing only the Zaireglycoprotein (FIG. 48A). Control animals received empty vectors (plasmidDNA and ADV-ΔE1 recombinant adenovirus), and vaccinated animals receivedthe multicomponent DNA vaccine containing NP and three subtypes of EbolaGP (pGP/NP), followed by ADV-GP(Z). As expected, anti-Ebola serumantibodies could not be detected in control animals, but in animalsreceiving the Ebola vaccine, an antigen-specific antibody response wasdetected at week 12, one month after the third DNA injection (FIG. 48B).After boosting with recombinant adenovirus, antibody titers increased10- to 20-fold over the levels obtained with DNA alone. Three monthsafter the final immunization, antibody levels remained high, except forone animal (subject 8) whose titer dropped slightly from 5×10⁴ to1.3×10⁴.

Primate cellular responses to Ebola antigens were next examined with anin vitro lymphocyte proliferation assay. In control monkeys,antigen-specific lymphocyte proliferation, measured by ³H-thymidineuptake, was equivalent to that in matched, unstimulated cells, resultingin a proliferation index near 1.0 for each animal (FIG. 48C). Incontrast, peripheral blood mononuclear cells (PBMC) from animalsimmunized with the multivalent vaccine showed 9- to 20-fold increasedstimulation, demonstrating a robust immune response to Ebola antigen atthe cellular level. Depletion of CD4-positive lymphocytes reduced theantigen-stimulated proliferative response of PBMC from vaccinatedmonkeys to the level observed in control animals (FIG. 48D). Depletionof CD8-positive lymphocytes, however, did not affect Ebolaantigen-specific lymphocyte proliferation. Therefore, the CD4-positivesubset of lymphocytes, which provide the T cell help required for highantibody titers, contributes to the vaccine-induced cellular immuneresponse.

To determine the protective efficacy of this vaccination regimen,monkeys were challenged with a lethal dose of the wild-type Mayingastrain from the Zaire subtype of Ebola virus. In the control monkeys,blood chemistry revealed an increase in hepatic enzymes (FIGS. 49A, B)that is characteristic for Ebola virus infection (Fisher-Hoch, S. P. etal. 1985 J Infect Dis 152:887-894). No such increase was observed invaccinated subjects. The elevation of serum alanine aminotransferase(ALT) and aspartate aminotransferase (AST) was parallel to a dramaticincrease in viraemia in all of the control animals (FIG. 49C). Incontrast, no substantial increase in viral load was observed invaccinated monkeys. The kinetics of disease progression was similaramong the control animals, and the disease incidence was 100% in thisgroup. Death occurred between days 5 and 6 for 3 animals, and the lastmonkey, moribund, was euthanized on day 7. In contrast, 4 out of 4monkeys immunized with the combination DNA-adenovirus vaccine survivedthis lethal challenge of Ebola virus, and sterilizing immunity wasachieved in 3 out of 4 subjects. The remaining animal showed a smalltransient rise in viral antigen; however, when followed long-term, allvaccinated animals showed no signs or symptoms of infection, and therewas no detectable viraemia for more than 6 months after infection, asmeasured by ELISA detection of viral antigen (FIG. 49A) and end pointtitration analysis of cultured virus. The vaccine recipient (subject 8)that exhibited a transient low level of viraemia on day 10 returned toundetectable levels by day 17.

As the natural reservoir for Ebola virus is unknown, the potential fortraditional public health measures to prevent future outbreaks islimited, thus increasing the urgency for the development of a vaccineand therapeutics in humans. The present findings demonstrate thatprimates can be immunized against the lethal effects of Ebola virusinfection, and that sterilizing immunity can be achieved using aheterologous prime-boost strategy. A multicomponent genetic vaccineexpressing Ebola virus structural proteins from diverse geographicisolates generated a strong antigen-specific immune response andresulted in the survival of immunized primates after challenge with alethal dose of Ebola Zaire, the subtype of this virus associated withthe highest number of deaths in human infections. The results of thisstudy suggest that T-cell mediated and humoral immunity contribute tovirus clearance in non-human primates, consistent with previous studiesin rodents (Xu, L. et al. 1998 Nat Med 4:37-42; Wilson, J. et al. 2000Science 287:1664-1666). Two immune parameters, antibody titer (1:75,000vs.<1:100, P=0.001) and the cellular proliferative response (˜12-foldvs. 1.4-fold, P=0.0014), provided highly significant immune correlatesof protection. Studies investigating the correlates of immune protectionfrom Ebola virus infection in humans are hampered by the aggressivenature of the virus and necessarily high level of biosafety containment.With the model of primate immunity presented here, it is envisioned asnow being possible to elucidate the mechanisms of immune protection fromEbola virus infection, to advance immune-based anti-viral therapies, andto develop a human vaccine for this pathogen and even other infectiouscauses of hemorrhagic fever.

Descriptions of Ebola, Marburg, and Lassa Constructs

VRC6000 VRC6000 (pVR1012-GP(Z)).

-   -   Backbone, pVR1012 (#450) expressing Ebola Glycoprotein of Zaire        Subtype. Orientation is BamHI/EcoRI/EcoRV/EcoRI/BglII)        VRC6001 VRC6001 (pVR1012x/s-GP(Z)) No other description.    -   This is the same as 6000, with the addition of an Sfi        restriction site to the pVR1012 backbone.        VRC6002 VRC6002 (pVR1012-GP(Z) delta MUC).    -   The mucin-like domain of GP(Z) was deleted. 530 bp in the        backbone, pVR1012 GP(Z) were deleted from EarI(2844) to        BfaI(3374). This mutant can bind to the Ebola receptor.        VRC6003 VRC6003 (pVR1012-GP(Z) delta MUC delta FUR).    -   The mucin-like domain and furin-cleavage site of GP(Z) were        deleted. 593 bp in the backbone, pVR1012 GP (Z) were deleted,        from EarI(2844) to EarI(3437). The protein has properties        similar to pVR1012-GP(Z) delta MUC.        VRC6004 VRC6004 (pVR1012-GP(Z) delta GP2).    -   A majority of the GP2 region in GP(Z) was deleted. 430 bp from        the backbone, pVR1012-GP (Z) were deleted from BclI(3414) to        BspEI(3844). The TM (transmembrane) region was retained.        VRC6005 VRC6005 (pVR1012-GP(Z) delta GP2 delta C-term A).    -   This is a C-terminal deletion of GP2. 267 bp were deleted from        the pVR1012-GP (Z) backbone, from MscI(3623) to BspMI(3890).        VRC6006 VRC6006 (pVR1012-GP(Z) delta GP2 delta C-term B).    -   This is a smaller deletion of GP2 C-terminal. 110 bp of backbone        pVR1012-GP(Z) were deleted from BstXI(3780) to BspMI(3890).        VRC6007 VRC6007 (pVR1012-GP(Z) delta GP2 delta FUS).    -   The fusion peptide in GP2 of GP(Z) was deleted in this mutant,        using PCR. 47 bp from the backbone, pVR1012-GP(Z), was deleted        from (3508-3555).        VRC6008 VRC6008 (pVR1012-GP(Z) delta TM).    -   The TM region of GP(Z) was truncated in this mutant. A stop        codon (TGA) was added downstream of the BspMI site(3889). This        protein is secreted and doesn't form a trimer.        VRC6052 VRC 6052 (pVR1012-GP(Z) delta sGP).    -   The majority of the SGP/GP homology region was deleted. 687 bp        from the backbone, pVR1012-GP(Z), were deleted from HincII(2083)        to HincII(2270).        VRC6101 VRC6101 (pVR1012x/s Ebola GP(R) (dTM)).    -   The vector expresses Ebola glycoprotein (subtype Reston) without        its transmembrane and intracellular domains. Using PCR, a stop        codon was generated downstream of a.a. 650 of GP(R), followed by        an XbaI site. This protein can be secreted and is termed        GP(R)(dTM).        VRC6110 VRC6110 (pAdApt Ebola GP(R) (dTM)).    -   An adenoviral shuttle vector expressing Ebola virus glycoprotein        (Reston subtype) without its transmembrane and intracellular        domains. Using PCR, a stop codon was generated downstream of        a.a. 651 of GP(Reston), followed by an XbaI site. The resulting        recombinant adenovirus expresses a 651a.a. secreted glycoprotein        termed GP(R)(dTM).        VRC6200 VRC6200 (pVR1012-GP(S)).    -   Backbone, pVR1012(#450), expressing Ebola Glycoprotein of the        Sudan Subtype. Orientation is        EcoRI/EcoRV/BamHI/BamHI/BamHI/XbaI.        VRC6201 VRC6201 (pVR1012x/s Ebola GP(S)).    -   No other description, but this is the same as 6200 with the        addition of an Sfi site to the 1012 backbone.        VRC6202 VRC6202 (pVR1012-GP(S) delta TM).    -   The TM region of GP(S) was truncated in this mutant. A stop        codon (TGA) was added downstream of the BspMI site(xxx). This        protein is secreted and doesn't form a trimer.        VRC6300 VRC6300 (pVR1012-GP(IC)).    -   Backbone, pVR1012(#450), expressing Ebola Glycoprotein of the        Ivory Coast Subtype. Orientation is        EcoRI/EcoRV/BamHI/BamHI/BamHI/XbaI.        VRC6301 VRC6301 (pVR1012x/s-GP(IC)).    -   No other description, but this is the same as 6300 with the        addition of an Sfi site to the 1012 backbone.        VRC6302 VRC6302 (pVR1012-GP(IC) delta TM).    -   The TM region of GP(IC) was truncated in this mutant. A stop        codon (TGA) was added downstream of the BspMI site. This protein        is secreted and doesn't form a trimer.        VRC6303 VRC 6303 (pVR1012x/s Ebola GP (IC) (dTM)).    -   A pVRC2000 based vector expressing Ebola glycoprotein (Ivory        Coast subtype) without transmembrane and intracellular domains.        Using PCR, a stop codon was generated downstream of a.a. 650,        followed by a BglII site. The vector expresses a 650 a.a.        secreted glycoprotein (a.a. 1-a.a. 650).        VRC6310 VRC6310 (pAdApt Ebola GP (IC) (dTM)).    -   An adenoviral shuttle vector expressing Ebola glycoprotein        (subtype Ivory Coast) without its transmembrane and        intracellular domains. Using PCR, a stop codon was generated        downstream of a.a. 651 of GP(IC). The resulting recombinant        adenovirus expresses a 651a.a secreted glycoprotein termed as        GP(IC)(dTM).        VRC6351 VRC6351 (pVR1012x/s-sGP(IC)). No other description.        VRC6400 VRC6400 (pVR1012-NP).    -   Backbone, pVR1012(#450) expressing Ebola Nucleoprotein of the        Ivory Coast Subtype.        VRC6401 VRC6401 (pVR1012x/s-NP).    -   No other description, but this is the same as 6400 with the        addition of an Sfi site to the 1012 backbone.        VRC6500 VRC6500 (pVR1012-VP35).    -   The backbone is pVR1012(#450). The insert is VP35 from Ebola        cloned from pGEM 3Zf(+)VP35(#1213).        VRC6600 VRC6600 (pAD/CMV-GP(dTM)(Z-CITE-S). No other        description.        VRC6601 VRC6601 (pAdApt Ebola GP(S)). No other description.        VRC6602 VRC6602 (pAdApt Ebola GP(S)(dTM)).    -   An adenoviral shuttle vector expressing Ebola glycoprotein        (Sudan subtype) without its transmembrane and intracellular        domains. A stop codon was fused downstream of a.a. 650 of GP(S).        The resulting recombinant adenovirus expresses a 654 a.a.        secreted glycoprotein, termed as GP(S)(dTM).        VRC6603 VRC6603 (pAdApt Ebola GP(Z)). No other description.        VRC6604 VRC6604 (pAdApt Ebola GP(Z)(dTM)).    -   Adenoviral shuttle vector expressing Ebola glycoprotein (subtype        Zaire) without its transmembrane and intracellular domains. A        stop codon was fused downstream of a.a. 651 of GP(Z). The        resulting recombinant adenovirus expresses a 655 a.a. secreted        glycoprotein termed as GP(Z)(dTM).        VRC6701 VRC6701 (pVR1012-Marburg).    -   Marburg glycoprotein (GP) open reading frame, Musoke strain.        Marburg was cloned into backbone #450(Bam(blunt)/XbaI) from        VRC6700 (Xba/PvuII).        VRC6702 VRC6702 (pVR1012x/s Marburg GP (dTM)).    -   This vector expresses the Marburg virus glycoprotein without its        transmembrane and intracellular domains. Using PCR, a stop codon        was generated downstream of a.a. 650 of GP(Marburg), followed by        a BglII site. This protein can be secreted and termed as        GP(Marburg)(dTM).        VRC6710 VRC6710 (pAdApt Marburg GP (dTM)).    -   Adenoviral shuttle vector (pVRC1290) expressing Marburg virus        glycoprotein without transmembrane and intracellular domains.        Using PCR, a terminator codon was generated downstream of a.a.        650, followed by a BglII site. The resulting recombinant        adenovirus expresses a 650 a.a. secreted protein (a.a. 1-a.a.        650).        VRC6800 VRC6800 (pVR1012x/s Lassa GP). No other description.        VRC6801 VRC6801 (pVR1012x/s Lassa GP (dTM). No other        description.        VRC6810 VRC6810 (pAdApt Lassa GP). No other description.        VRC6811 VRC6811 (pAdApt Lassa GP (dTM)). No other description.

EXAMPLE 1

Vector construction. The construction of DNA vectors expressing EbolaZaire glycoprotein (GP), secreted GP (SGP), and nucleoprotein (NP) hasbeen described in Xu, L. et al. 1998 Nat Med 4:37-42. The GP Sudan andIvory Coast expression vectors were constructed similarly. Briefly, GPopen reading frames were generated from polymerase chain reaction afterreverse transcription of RNA (RT-PCR) products of infected cell RNAusing the following primers: 5′ ATC TTC AGG ATC TCG CCA TGG A 3′ (SudanGP gene; NcoI>ATG; SEQ ID NO: 44), 5′ GAT ATT CAA CAA AGC AGC TTG CAG 3′(Sudan GP gene; C-terminus GP stop; SEQ ID NO: 45), 5′ CTA ATC ACA GTCACC ATG GGA 3′ (Ivory Coast GP gene; NcoI>ATG; SEQ ID NO: 46), 5′ AAAGTA TGA TGC TAT ATT AGT TCA 3′ (Ivory Coast GP gene; C-terminus GP stop;SEQ ID NO: 47) yielding the TA clones PCR2.1 Sudan and PCR2.1 IvoryCoast. The Sudan glycoprotein was digested from plasmid PCR2.1 withXbaI/HindIII, Klenow treated, and cloned into the XbaI site of p1012(Xu, L. et al. 1998 Nat Med 4:37-42). Ivory Coast GP was digested fromplasmid PCR2.1 with EcoRI, Klenow treated, and cloned into the XbaI siteof p1012 (Xu, L. et al. 1998 Nat Med 4:37-42).

To make ADV-GP, the BamHI/EcoRI fragment of GP(Z) was digested frompGEM-3Zf(−)-GP, treated with Klenow, and inserted intoHindIII/XbaI/Kle/CIP treated pRc/CMV plasmid. The resulting plasmid(PRC/CMV-GP(Z)) was digested by NruI/DraIII and treated with Klenow. TheNruI/DraIII/Kle fragment containing the CMV enhancer, GP(Z) DNA andbovine growth hormone polyadenylation signal was inserted into the BglIIsite of the adenoviral shuttle plasmid pAdBgIII (Ohno, T. et al. 1994Science 265:781-784). The adenovirus, a first generation dl 309-basedAd5 vector, contained a deletion in E1 to render the vectorreplication-defective and a partial deletion/substitution in E3, whichdisrupts the coding sequences for the E3 proteins with a relativemolecular mass of 14.7 kD, 14.5 kD and 10.4 kD, respectively. Therecombinant adenovirus expressing Zaire GP, ADV-GP(Z), was madeaccording to previously published methods (Aoki, K. et al. 1999 Mol Med5:224-231). The dose of adenovirus administered, 10¹⁰ plaque-formingunits (PFU) per animal (approximately 3×10⁹ PFU/kg), is within the rangeused safely in human gene therapy trials.

Animal study and safety. Eight cynomolgus macaques (Macacafascicularis), 3 years of age and weighing 2-3 kg, obtained from Covance(Princeton, N.J.), were used for the immunization and challengeexperiment. To obtain blood specimens and administer vaccines, themonkeys were anesthetized with Ketamine. The animals were housed singlyand received regular enrichment according to the Guide for the Care andUse of Laboratory Animals (DHEW No. NIH 86-23). Just before the Ebolavirus challenge and up to the end of the experiment, the animals weremaintained in the Maximum Containment Laboratory (BSL-4) and fed andchecked daily. One animal was euthanized that appeared moribund and wassubsequently necropsied for pathologic examination. In addition, asingle asymptomatic vaccinated animal was euthanized for pathologic andvirologic analysis.

Mouse immunization. DNA and adenovirus vectors expressing Ebola Zaire GPor NP were constructed as described previously (Xu, L. et al. 1998 NatMed 4:37-42; Ohno, T. et al. 1994 Science 265:781-784), with geneexpression under the control of the cytomegalovirus enhancer andpromoter. Mice were immunized intramuscularly with 100 μg of DNA (pGP ora p1012 plasmid control) or 10⁸ PFU of adenovirus (ADV-GP or ADV-ΔE1control virus) on days 0, 14, and 28 and blood was collected on day 28.On day 42, mice received an intramuscular boost with DNA or adenovirusand titers were re-measured on day 56. ELISA IgG titers were determinedusing 96-well plates coated with a preparation of Ebola virus antigenderived from purified virions and enriched for membrane-associatedproteins (GP, VP40 and VP24) (Ksiazek, T. G. et al. 1992 J ClinMicrobiol 30:947-950). Specific antigen binding was detected using agoat anti-human IgG(H+L)-horseradish peroxidase conjugate andABTS/Peroxide (substrate/indicator).

Macaque immunization. For the DNA immunizations, animals received 1 mgeach of DNA expressing GP(Zaire) [GP(Z)], GP(Ivory Coast) [pGP(IC)],GP(Sudan) [pGP(S)] and NP(Zaire) administered as a mixture [pGP/NP], or4 mg empty [pGP(Z)] control plasmid bilaterally (2 mg per side) in thedeltoid muscle. Immunization at weeks 0 and 4 were by IM injection, andat week 8 by Biojector. For the adenovirus boost, animals received 10¹⁰PFU of ADV-GP (Zaire subtype) or ADV-ΔE1 (empty vector) divided into twodoses administered bilaterally in the deltoid muscle. At week 32, allanimals received an intraperitoneal injection of approximately 6 PFUs ofEbola virus (Zaire 1976 isolate; Maying a strain) (Kiley, M. P. et al.1980 J Gen Virol 49:333-341) in 1 ml Hanks' buffered salt solution. Thevirus was isolated directly from patient blood and used after a singlepassage in Vero cells.

ELISA IgG titers were determined as above for control (Plasmid: ADV-ΔE1)and immunized [pGP/NP: ADV-GP(Z)] monkeys. The reciprocal endpoint ofdilution for each subject was at week 12 and week 24. Serum antibodylevels were measured by ELISA as described (Ksiazek, T. G. et al. 1992 JClin Microbiol 30:947-950).

Blood was collected from control (plasmid: ADV-ΔE1) or immunized[pGP/NP: ADV-GP(Z)] animals 1-3 days prior to the immunizations at weeks4, 8 and 20, and at week 24. Blood was separated over a Percoll gradientto obtain the lymphocyte enriched population. Lymphocytes werestimulated as described (Xu, L. et al. 1998 Nat Med 4:37-42) for 5 daysin vitro using supernatant from cells transfected with either Ebolasecreted glycoprotein (SGP) or empty plasmid, and proliferation wasmeasured by ³H-thymidine uptake. The proliferation index was calculatedas the proliferation in wells receiving SGP divided by proliferation inwells receiving control supernatant.

Viral detection in macaques. The presence of circulating Ebola virusantigen was detected as described (Ksiazek, T. G. et al. 1992 J ClinMicrobiol 30:947-950) by capturing VP40 protein from serial dilutions ofmonkey plasma. 96-well plates coated with antiVP40 mAb were used tocapture antigen, and detection was with a rabbit anti-Ebola virus serum.

EXAMPLE 2

The amino acid sequences of Ebola GP(Zaire) and NP (Zaire) were obtainedfrom Genbank: GP(Zaire), Genbank accession no. P87666; NP(Zaire),Genbank accession no. NC_(—)002549; while GP(Sudan/Gulu) was obtainedfrom the CDC. The amino acid sequences were then back-translated to DNAsequences using mammalian preferred codons. Serial 75 bp oligos with 25bp overlapping were prepared to cover the entire gene. The oligos werethen assembled into intact mammalian genes containing preferred codonsusing PCR. In the design, a stop codon was introduced in front of thepredicted transmembrane domains of GP(Zaire) (a.a. 648-676) and GP(Sudan/Gulu) (a.a. 648-676) so that this region was excluded from thesesynthetically created genes. The deletions also led to the loss of a 4a.a. cytoplasmic region in both constructs. Final sequencing of theEbola GP (Zaire) sequence revealed 10 divergent amino acids from thelaboratory GP sequence, which was used in our animal studies and thesewere corrected by site-directed mutagenesis. These inserts were clonedinto p1012 x/s by XbaI/Sa/I.

Construction of CMV/R-GP(S/G)(ΔTM)/h

The codon-modified, transmembrane domain deleted form of the Ebola GP(Sudan/Gulu) gene was excised from p1012 (x/s)-GP(S/G)(ΔTM)/h usingSa/I/KpnI, and inserted into the Sa/I/KpnI digested CMV/R/MCS plasmid.

Construction of CMV/R GP(Z) (ΔTM)/h

The codon-modified, transmembrane domain deleted form of the Ebola GP(Zaire) gene was excised from p1012 x/s-GP (Z)(dTM)/h Sa/I/BglII sitesand cloned into the Sa/I/BglII sites of the CMV/R plasmid.

Construction of CMV/R Ebola NP

The NotI-KpnI fragment from VRC6400 (pVR1012-NP) expressing Ebolanucleoprotein of Zaire Subtype was excised and cloned into the NotI/KpnIsites of the CMV/R plasmid.

EXAMPLE 3 Improved Non-Viral Mammalian Expression Vector

This invention provides an improved mammalian expression vector whichgenerates a higher level of protein expression than vectors currently inuse.

Initially, 3 new vectors, each containing a different enhancer, weredeveloped and tested. The RSV enhancer, the mouse ubiquitin enhancer(mUBB), and the CMV enhancer (Xu et al. 1998 Nature Med. 4:37-42) wereeach combined with the HTLV-1 R region (Takebe et al. 1988 Mol Cell Biol8:466-472) to create separate vectors. When these 3 vectors werecompared to the backbone containing the CMV enhancer in combination withthe CMV translational enhancer and intron (CMVint), which is currentlythe most effective vector, in vitro data showed that expression with thevector containing the CMV/R was increased 5-10 fold compared to CMV/int,and immunological studies showed induction of significantly higher CD4and CD8 T cell responses compared to CMVint. Both in vivo and in vitroresponses were markedly higher with this new vector. Neither of theother two vectors produced comparable results.

The expression vector is unique in that it uses a specific translationalenhancer in combination with specific enhancer/promoters to yield highlevels of expression and enhanced immunogenicity for DNA vaccines. Thisis particularly important because the potency of these vaccines inhumans is marginal and generic improvements can serve as importantplatforms to make the technology practical for human use. The expressionvector cassettes can be used in other gene based vaccines as well, orfor production of recombinant proteins from eukaryotic expressionvectors. The invention is useful in the production of genetic vaccinesand gene therapies for a wide variety of diseases, including HIV andother viral diseases and cancer.

FIG. 50. Enhanced Expression of Modified CMV Expression Vector, CMV/R.

Mouse fibroblast 3T3 cells were transfected with (A) vector alone (lane1), CMVint-gp-145(dCFI) (lane 2), CMV/R-gp145(dCFI) (lane 3) or (B)mUBB-gp145(dCFI) (lane 4), mUBB/R-gp145(dCFI) (lane 5) in 6-well tissueculture dishes with 0.5 ug of the corresponding plasmids using calciumphosphate. 24 hours after transfection, cells were harvested and lysedin lysis buffer (50 mM HEPES, 150 mM NaCl, 1% NP-40, Mini Completeprotease inhibitor cocktail (Roche)). 10 μg of total protein of eachsample were separated on a 4-15% gradient gel using SDS-PAGE, followedby protein transfer and Western blot analysis. Human HIV-IgG (1:5000)was used as the primary antibody, and HRP-conjugated goat anti-human IgG(1:5000) as the secondary antibody. The membrane was developed using theECL Western blot developing system. The arrow indicates the specificband for the HIV Env gp145(ΔCFI) polyprotein.

FIG. 51. Enhanced Immunogenicity of Modified CMV Expression Vector,CMV/R, in Mice.

Five mice in each group were immunized with 50 μg of the indicatedplasmid DNA at weeks 0, 2, and 6. 10 days after the last injection,splenocytes from each mouse were harvested and stimulated using a poolof control peptides (15 mer), or a pool of HIV Env peptides (15 mer) for6 hours. The stimulated splenocytes were stained using a cocktail ofantibodies containing PE-anti-mouse CD3, PerCP-anti-mouse CD4,APC-anti-mouse CD8, FITC-anti-mouse IFN-γ and FITC-anti-mouse TNF-α. Thesamples were analyzed by flow cytometry. CD3/CD4/IFN-γ/TNF-α andCD3/CD8/IFN-γ/TNF-α positive cell numbers were measured using FloJosoftware (Treestar).

The CMV Enhancer/Promoter, R Region (HTVL-1), CMV IE Splicing AcceptorSequence

(SEQ ID NO: 52): CCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCATCGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCTTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTACGTCCGCCGTCTAGGTAAGTTTAGAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTAGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCT TTTCTGCAG1-741: CMV Enhancer/Promoter742-972: HTLV-1 R region973-1095: CMV/IE Splicing Acceptor

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications and publications referredto above are hereby incorporated by reference.

What is claimed is:
 1. A vaccine comprising an adenoviral vectorcomprising a sequence encoding Marburg virus glycoprotein being at least95% identical to Marburg virus glycoprotein that is encoded in theconstruct VRC6701 (SEQ ID NO:30).
 2. The vaccine of claim 1, wherein thesequence encoding Marburg virus glycoprotein is the sequence as presentin the construct VRC6701 (SEQ ID NO:30).
 3. A composition for boostingan immune response to a viral antigen in an individual, comprising anadenoviral vector comprising a sequence encoding Marburg virusglycoprotein being at least 95% identical to Marburg virus glycoproteinthat is encoded in the construct VRC6701 (SEQ ID NO:30).
 4. Thecomposition of claim 3, wherein the sequence encoding Marburg virusglycoprotein is the sequence as present in construct VRC6701 (SEQ IDNO:30).
 5. A method for boosting an immune response to a viral antigenin an individual, comprising administering to the individual acomposition comprising an adenoviral vector comprising a sequenceencoding Marburg virus glycoprotein being at least 95% identical toMarburg virus glycoprotein that is encoded in the construct VRC6701 (SEQID NO:30).
 6. The method of claim 5, wherein the sequence encodingMarburg virus glycoprotein is the sequence as present in VRC6701 (SEQ IDNO:30).
 7. The method of claim 5, wherein the viral antigen is a Marburgvirus antigen.
 8. The method of claim 5, wherein the administering isperformed by injection.
 9. The method of claim 8, wherein theadministering is performed at a dose of 5×10⁷ to 1×10¹² particles perinjection.